In addition to the specificity andefficiency of an siRNA sequencefor a target gene which are ofhigh importance in post-transcriptionalgene silencing (PTGS) thedelivery of siRNA into cells is crucial.An efficient method to optimizetransfection and gene silencin

In addition to the specificity and
efficiency of an siRNA sequence
for a target gene, which are of
high importance in post-transcriptional
gene silencing (PTGS), the
delivery of siRNA into cells is crucial.
An efficient method to optimize
transfection and gene silencing
is critical for successful RNAi
experiments. In this Application
Note HeLaS3 cells were transfected
with GFP expressing plasmid
and Cy5-labeled siRNA against
GFP, both individually and in
combination, using QIAGEN
transfection reagents. Uptake of
labeled siRNA and the gene
silencing effect were monitored
for up to 24 hours after transfection.
Efficient gene silencing was
achieved which confirms the
operation of RNAi mechanism in
the selected cell line.
The technique of RNAi has the
potential to revolutionize functional
genomics. The pathways and
mechanisms of RNAi are currently
under intense investigation, and a
good understanding of these is
essential to allow scientists to
exploit the applications of RNAi.
Simple and efficient methods for
monitoring gene silencing are
important tools for RNAi experiments.
In this Application Note we
describe the evaluation of a
method using fluorescent labels to
determine transfection efficiency
and the gene silencing effect.
HeLaS3 cells were transfected
with siRNA using RNAiFect
Reagent. TransMessenger
Reagent was used for the transfection
of GFP expressing plasmid (pGFP). Cells were also co-transfected
with both plasmid DNA and
siRNA using TransMessenger
Reagent. The uptake of siRNA into
mammalian cells was monitored
using fluorescent labeled siRNA
and the gene silencing effect and
level of cellular protein expression
were quantified at 4, 8, and 24
hours after transfection.
Materials and methods
HeLa S3 cells( ATCC, CCL-2.2)
were plated at 5 x 104 cells /well
in 24-well plates and incubated for
24 hours before transfection. Cells
were transfected with 0.9 g of
GFP22-siRNA (QIAGEN, Cat. #
1022064) per well using RNAiFect
Reagent (QIAGEN, Cat. #301605).
GFP22-siRNA has a homologous
sequence to GFP mRNA. It was
transfected unlabeled or labeled with Cy5. For GFP expression,
cells were transfected with 0.2 g
of pGFP in each well using Trans-
Messenger Reagent (QIAGEN, Cat.
# 301525). For co-transfection of
GFP22-siRNA and pGFP, Trans-
Messenger Reagent was also used.
The recommended protocols,
described in the QIAGEN RNAi-
Fect handbook and the supplementary
protocol for co-transfection
of adherent cells with siRNA
and plasmid DNA using TansMessenger
Reagent was followed,
using the optimized ratio of RNAi-
Fect Reagent : siRNA and Trans-
Messenger Reagent : siRNA of 1:5
as determined previously.
Analysis of GFP expression and
Cy5 fluorescence was performed
using the Agilent 2100 bioanalyzer
with pressure cartridge for flow
cytometric studies.
For the analysis with the Agilent
2100 bioanalyzer, cells were
trypsinized and washed twice in
phosphate buffered saline (PBS),
and then resuspended in Cell
Buffer (CB) at a dilution of 1 x 106
-1.5 x 106 per milliliter. Cells were
stained for 30 minutes at RT with
the live dyes calcein-AM (Molecular
Probes, Cat. # C-3099) or carboxynaphthofluorescein
diacetate(
CBNF) (Molecular Probes,
Cat. # C-13196). Dye concentrations
of 2.5 M were used for
staining. Ten microliters of each
sample were loaded on a microfluidic
chip for analysis. Microfluidic
chips and CB buffer were supplied
with the Agilent Cell Fluorescence
LabChip kit. Cy5 fluorescence
indicated the efficiency of siRNA
uptake and calcein-AM staining
monitored the cell viabilit
y. In cells transfected with pGFP, GFP
fluorescence indicated the transfection
efficiency and the proportion
of living cells was monitored
by CBNF staining. The efficiency
of gene silencing in cells co-transfected
with Cy5-siRNA and pGFP
was monitored by GFP fluorescence,
and the viability of the cells
was monitored by CBNF staining.
Fluorescence microscopy was
used to compare the 2100 bioanalyzer
results 24 hours after transfection.Results and discussion

Cells were analyzed 4, 8, and 24
hours after transfection using the
2100 bioanalyzer. As controls,
untransfected cells were used,
either unstained, stained with calcein-
AM or CBNF alone. Controls
were analyzed in parallel with the
transfected cells (data not shown).
In cells transfected with Cy5-
labeled siRNA, Cy5 positive cells
were detectable after 4 hours (figure
1). Transfection efficiency
measurements after calcein staining
showed that over 80 % of the
live cells were Cy5-positive. This
picture remained constant after 24
hours. In cells transfected with
pGFP, the proportions of live cells
expressing GFP increased from
22.4 % after 4 hours to 57.6 % after
24 hours. Co-transfection of pGFP
and siRNA resulted in efficient
gene silencing and results after
each time point were compared
and plotted (figure 2). A strong
gene silencing effect was measured
and by 24 hours the measured
down-regulation of GFP
expression was up to 77 %.

In a separate experiment, cells
were transfected with pGFP alone,
pGFP with unlabeled siRNA, and pGFP with Cy5-labeled siRNA to
determine whether the Cy5 label
affected gene silencing. Results,
24 hours after transfection,
showed similar down regulation
of the GFP expression, meaning
that the Cy5 label did not interfere
significantly with the gene silenci
ng
effect (figure 3).

Cells were
also examined by fluorescence
microscopy confirming the results
from the 2100 bioanalyzer for GFP
existence in cells (figure 4).

Conclusions
Uptake of labeled siRNA and its
gene silencing effect were quantitatively
monitored using the flow
cytometry capability of the Agilent
2100 bioanalyzer. Successful
co-transfection of pGFP and
Cy5-labeled siRNA using Trans-
Messenger reagent was achieved.
GFP expression of up to 56.9 %,
and silencing of GFP expression
to levels below 10 % confirmed
the efficient transfection and
silencing mechanism in the selected
cell line.
The measurement of protein
expression in viable cells with the
Agilent 2100 bioanalyzer is fast,
accurate and automated, providing
efficient methods to monitor
and optimize any gene silencing
experiment. QIAGEN reagents
and chemically synthesized siRNA
probes are highly efficient and
reliable.

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